Viral RNA Mini Kit Research Articles

Longitudinal wastewater-based surveillance of SARS-CoV-2 during 2023 in Ethiopia.

Although wastewater-based epidemiology (WBE) successfully functioned as a tool for monitoring the coronavirus disease 2019 (COVID-19) pandemic globally, relatively little is known about its utility in low-income countries. This study aimed to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater, estimate the number of infected individuals in the catchment areas, and correlate the results with the clinically reported COVID-19 cases in Addis Ababa, Ethiopia. A total of 323 influent and 33 effluent wastewater samples were collected from three Wastewater Treatment Plants (WWTPs) using a 24-h composite Moore swab sampling method from February to November 2023. The virus was captured using Ceres Nanotrap® Enhancement Reagent 2 and Nanotrap® Microbiome A Particles, and then nucleic acids were extracted using the Qiagen QIAamp Viral RNA Mini Kit. The ThermoFisher TaqPath™ COVID-19 kit was applied to perform real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to quantify the SARS-CoV-2 RNA. Wastewater viral concentrations were normalized using flow rate and number of people served. In the sampling period, spearman correlation was used to compare the SARS-CoV-2 target gene concentration to the reported COVID-19 cases. The numbers of infected individuals under each treatment plant were calculated considering the target genes' concentration, the flow rate of treatment plants, a gram of feces per person-day, and RNA copies per gram of feces. SARS-CoV-2 was detected in 94% of untreated wastewater samples. All effluent wastewater samples (n = 22) from the upflow anaerobic sludge blanket (UASB) reactor and membrane bioreactor (MBR) technology were SARS-COV-2 RNA negative. In contrast, two out of 11 effluents from Waste Stabilization Pond were found positive. Positive correlations were observed between the weekly average SARS-CoV-2 concentration and the cumulative weekly reported COVID-19 cases in Addis Ababa. The estimated number of infected people in the Kality Treatment catchment area was 330 times the number of COVID-19 cases reported during the study period in Addis Ababa. This study revealed that SARS-CoV-2 was circulating in the community and confirmed previous reports of more asymptomatic COVID-19 cases in Ethiopia. Additionally, this study provides further evidence of the importance of wastewater-based surveillance in general to monitor infectious diseases in low-income settings. Wastewater-based surveillance of SARS-CoV-2 can be a useful method for tracking the increment of COVID-19 cases before it spreads widely throughout the community.

Predominance of DENV-3 among patients in Ouagadougou, Burkina Faso.

Dengue is an emerging vector-borne viral disease in tropical and subtropical areas such as Burkina Faso that experienced dengue outbreak in, 2013, 2016, 2017 and more recently in 2023. This study aimed to determine the seroprevalence and dengue serotype in suspected patients in Ouagadougou, Burkina Faso. The study was conducted during October and November 2023 and included suspected febrile patients seen at HOSCO and CERBA. Plasma or serum samples were used for the detection of non-structural proteins (NS1) and IgM and IgG antibodies against the dengue virus using SD Bioline Dengue Duo rapid detection kit. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit and dengue serotypes were determined by real-time RT-PCR using the Dengue Real-TM Genotype kit. The study population consisted of 896 patients, including 397 (44.3%) men and 499 (55.7%) women. Dengue seroprevalence was 16.5% (148/896) with 14.1% (126/896) of patients positive for the NS1 antigen, 1.3% (12/896) positive for IgM, and 2.7% (24/896) positive for IgG. Serotyping among 40 out of 45 positive patients revealed 77.5% (31/40) DENV-3, 17.5% (7/40) DENV-1, and 5.0% (2/40) DENV-2. The present study report a high seroprevalence of dengue virus infection among patients during the months considered as the peak of infection in Burkina Faso. The results revealed a predominance of DENV-3. Continuous surveillance of dengue virus serotypes circulating in Burkina Faso is crucial.

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

First detection of Rift Valley Fever Virus among Culex pipiens in Tahoua, Niger

The Rift Valley Fever (RVF) is an arbovirus disease responsible of regular epizootics and epidemics in sub-Saharan Africa and Arabian Peninsula. In 2016, Niger experienced its first outbreak of RVF in Tahoua region, which resulted in high consequences in animal and human health. The aim of this study was to investigate on the RVFV circulation among potential vectors of the disease. This was a cross-sectional survey carried out in Tahoua and Agadez regions in August 2021. Adult mosquitoes were collected by using the morning spray in human dwellings and the CDC light trap methods. After morphological identification, viral RNA was extracted. The RNA was extracted by using QIAamp Viral RNA Mini Kit (Qiagen). The RVFV detection was performed by using the qRT-PCR method. A total of 2487 insects (1978 mosquitoes, 509 sandflies and 251 biting midges) were identified and divided into three families (Culicidae, Psychodidae and Ceratopogonidae). The Culicidae family composed of the Culex genus being the most abundant with a predominance of Cx.pipiens (31.88%; n = 793) followed by Mansonia sp (21.51%; n = 535), Anophelesgambiae s.l. (8.44%; n = 210), An. pharoensis (0.72%; n = 18), An. rufipes (0.48%; n = 12), Cx. quinquefasciatus (6.39%; n = 159), the Psychodidae with sandflies (20.46%; n = 509), and the Ceratopogonidae with Culicoides genus (10.09%; n = 251). The qRT-PCR carried out on a sample of mosquitoes (N = 96) highlighted that one individual of Cx.pipiens was found positive to RVFV. This specimen was from Tassara locality (Tahoua) and collected by CDC Light Trap method. This study reveals for the first time the circulation of RVFV among Cx.pipiens in Niger and highlights the possible vectorial role of this vector in the disease transmission. Further investigations should be carried out to identify the biological and ecological determinants that support the maintenance of the virus in this area in order to guide control interventions.

RT-qPCR investigation of post-mortem tissues during COVID-19.

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.

Evaluation of the time, labor, and money required for manual and automated nucleic acid (RNA) isolation for the detection of SARS-COV-2 by QRT-PCR using the qiamp viral RNA mini kit and kingfisher flex

We undertook this cross-sectional investigation to assess the time, manpower, and average run cost per sample using manual Qiamp Viral RNA micro kit (Qiagen) and automated kingfisher flex instrument extraction methods for SARS-Cov-2 identification.The study used 120 Viral Transport Media-collected nasopharyngeal/ oropharyngeal swabs.Magnetic bead-based RNA extraction was performed using the Thermo Fisher Scientific kingfisher flex instrument and manual Extraction was Silica membrane-based Qiagen spin column kits. The TaqPath™ COVID-19 Combo Kit from Thermo Fisher Scientific was used for detecting SARS-CoV-2 target genes.Human technique took 40 minutes longer than automation. It cost more to automate than to manually labor. These disparities in time, effort, and cost affect laboratory operations, offering pros and cons for each method. This suggests that positive or negative was consistent regardless of viral load or RNA concentration.The study found that automated RNA extraction yielded better results compared to manual extraction. The automated sample processing system saved time, people, and money. In resource-limited or low-throughput labs, manual extraction may be preferable. Manual methods are laborious, require more hands-on time, and risk cross-contamination and technical blunders.

Microbiome Sex-Related Diversity in Non-Muscle-Invasive Urothelial Bladder Cancer.

Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors.

First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid–liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2.•Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Phylogenetic, Sequencing, and Mutation Analysis of SARS-CoV-2 Omicron (BA.1) and Its Subvariants (BA.1.1, BA.2) During the Fifth Wave of the COVID-19 Pandemic in the Iraqi Kurdistan Region.

Introduction In December 2019, a global outbreak of SARS-CoV-2 occurred in Wuhan, China, resulting in the COVID-19 pandemic. Since then, the virus has spread to all countries, necessitating a worldwide initiative to create effective treatments and vaccines. Methods The RNA of samples QIAamp Viral RNA Mini Kit (Qiagen, MD).SARS-CoV-2 RNA was reverse transcribed with SuperScript IV VILO (ThermoFisher Scientific, Waltham, MA). The virus cDNA was amplified in two multiplexed PCR reactions using Q5 DNA High-fidelity Polymerase (New England Biolabs, Ipswich, MA). The genome was entirely sequenced from 40 samples at the Scripps Research Institute (TSRI) in California, USA. The samples were sequenced using a NovaSeq 6000 SP Reagent Kit v1.5 (Illumina, USA). The TSRI then entered these sequences into the GISAID database. The virus sequence was matched to the SARS-COV-2 virus identified in Wuhan, China (accession number: NC 045512.2) using Illumina sequencing technology (Illumina, CA), finding 95 different changes. The NextClade (clades.nextstrain.org) and Mega 11 (https://www.megasoftware.net) software tools were used to analyze SARS-CoV-2 genome sequence alignment and mutation studies. Results Following a sequencing analysis, it was determined that the spike glycoprotein (S) included a total of 38 mutations. Thirty of these mutations were found in the ORF1a gene. Additionally, 11 mutations were found in the ORF1b gene, with the remaining mutations found in the nucleocapsid (N), membrane protein (M), open reading frames 6 (ORF6), open reading frames 9 (ORF9), and envelope (E) genes. The phylogenetic analysis and transmission studies indicated that the isolates discovered in Iraq had separate infection origins and were closely linked to those discovered in other nations and states. Conclusion According to the findings of this study, a new vaccine can be developed based on identifying new Omicron variant mutations and subvariants such as BA.2, which were identified for the first time in Iraq.

Analysis of RdRp Gene in SARS CoV-2 Variants

SARS CoV-2 was discovered in Wuhan, China, in December 2019 and has since mutated into new variants. One of the genes that mutated in SARS CoV-2 is RNA-dependent RNA polymerase (RdRp), a gene playing roles in viral replication and transcription. This study aimed to determine the variation of the RdRp gene in the SARS CoV-2 variant. We used VTM-preserved biological samples from positive nasopharyngeal swabs confirmed by qRT-PCR testing stored at -80°C. We isolated samples using the QIAamp Viral RNA Mini Kit and determined their concentration with Qubit RNA HS Assay reagents. After amplifying the RNA using qRT-PCR, we prepared the samples for sequencing with the Illumina RNA prep kit. Sequencing was carried out with the Miseq system and results were produced in FASTQ format. This study identified 251 samples, and analysis of the RdRp gene showed 69 substitution mutations and one deletion mutation. The highest point mutation was found in the C14408T nucleotide sequence, almost in all study samples of all variants (98.4%). There were 28 missense mutations, including C14408T, which converts the amino acid proline to leucine. Four research samples that did not have mutations in C14408T were samples in the early period of the COVID-19 pandemic before various variants were reported. In summary, our study on the variation of the RdRp gene in the SARS CoV-2 variant demonstrated remarkable C14408T mutations. Utilizing this mutation is a crucial genetic indicator for tracking alterations in the SARS-CoV-2 and comprehending its evolutionary patterns. Keywords: SARS CoV-2, COVID-19, variant, mutation, RdRp gene

Molecular surveillance of arboviruses in Nigeria

Arboviral infections are fast becoming a global public health concern as a result of its high fatality rate and sporadic spread. From the outbreak of Zika virus in the Americas, the endemicity of Yellow fever in West Africa and South America, outbreaks of West Nile virus in South Africa to the year-round and national risk of Dengue fever in Mainland China and India. The war against emerging and re-emerging viral infection could probably lead to the next pandemic. To be above the pending possible arboviral pandemic, consistent surveillance of these pathogens is necessary in every society. This study was aimed at conducting a surveillance for Yellow fever virus, Zika virus, Chikungunya virus, Dengue virus and Rift Valley fever virus in four states in Nigeria using molecular techniques. A cross-sectional study involving 1600 blood samples collected from febrile patients in Lagos, Kwara, Ondo and Delta States between 2018 and 2021 was conducted using Real time polymerase chain reaction for detection of the pathogens. Extraction and purification of viral RNA were done using Qiagen Viral RNA Mini Kit. Samples were analyzed using One Step PrimeScript III RT-PCR mix (Takara Bio) alongside optimized primers and probes designed in-house. Positive samples were sequenced on MinION platform (Nanopore technologies). Bioinformatic and phylogenetic analysis were performed with DNASTAR Lasergene 17.3. All the RNA extracted from samples collected from the four states were negative for ZIKV RNA, RVFV RNA, CHIKV RNA and DENV RNA. However, twelve of the samples (2%) tested positive for YFV RNA. Three full genomes of sizes 10,751 bp, 10,500 bp and 10,715 bp were generated and deposited in GenBank with accession numbers: ON323052, ON323053 and ON323054 respectively. Phylogenetic analysis shows clustering within lineage 3 of West African genotype. This result shows an active spread of Yellow fever in Delta State, Nigeria. However, there is no emergence of a new genotype There is a need for an intense surveillance of Yellow fever virus in Nigeria to avert a major outbreak.

A Comprehensive Phylogenetic Analysis of SARS-CoV-2: Utilizing a Novel and Convenient In-House RT-PCR Method for Characterization without Virus Culture and BSL-3 Facilities.

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14-685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.

Sequencing the SARS-CoV-2 Genome from Stool Samples of Post-acute Cases Implicates a Novel Mutation Associated with Reduced Antibody Neutralization.

Whole-genome SARS-CoV-2 sequencing tools are crucial for tracking the COVID-19 pandemic. However, current techniques require sampling of actively infectious patients following COVID-19 testing to recover enough SARS-CoV-2 RNA from the nasopharyngeal passage, which rapidly clears during the first few weeks of infection. A prospective assessment of the viral genome sourced from recovered non-infectious patients would greatly facilitate epidemiological tracking. Thus, we developed a protocol to isolate and sequence the genome of SARS-CoV-2 from stool samples of post-acute SARS-CoV-2 patients, at timepoints ranging from 10-120 days after onset of symptoms. Stool samples were collected from patients at varying timepoints post-convalescence, and viral DNA was isolated and sequenced using the QIAamp Viral RNA Mini Kit (Qiagen Inc.) and Ion Ampliseq™ Library Kit Plus (Life Technologies Corporation). Capacity of neutralizing antibodies in patient plasma was tested using a Luminex panel (Coronavirus Ig Total Human 11-Plex ProcartaPlex™ Panel, ThermoFisher). Of 64 samples obtained from post-acute patients, 21 (32.8%) yielded sufficient material for whole-genome sequencing. This allowed us to identify widely divergent phylogenetic relativity of the SARS-CoV-2 genome from post-acute patients living in the same households and infected around the same time. Additionally, we observed that individuals who recovered from infection expressed varying degrees of antibodies against SARS-CoV-2 structural proteins that corresponded to distinct variants. Interestingly, we identified a novel point mutation in the viral genome where infected patients expressed antibodies with a significantly reduced capacity to neutralize the virus in vitro relative to that of those infected with the wild-type strain. Altogether, we demonstrate a protocol to successfully sequence the SARS-CoV-2 genome from stool samples from patients up to 4 months post-infection, which can be applied to studies that assess the relationship between variants and immune response post-hoc and safe monitoring of the SARS-CoV-2 genome during the pandemic.

Is SARS-CoV-2 a concern in the largest wastewater treatment plant in middle east?

The surveillance of wastewater treatment plant (WWTP) as the end point of SARS-CoV-2 shed from infected people arise a speculation on transmission of this virus of concern from WWTP in epidemic period. To this end, the present study was developed to comprehensively investigate the presence of SARS-CoV-2 in raw wastewater, effluent and air inhaled by workers and employee in the largest WWTP in Tehran for one-year study period. The monthly raw wastewater, effluent and air samples of WWTP were taken and the SARS-CoV-2 RNA were detected using QIAamp Viral RNA Mini Kit and real-time RT-PCR assay. According to results, the speculation on the presence of SARS-CoV-2 was proved in WWTP by detection this virus in raw wastewater. However, no SARS-CoV-2 was found in both effluent and air of WWTP; this presents the low or no infection for workers and employee in WWTP. Furthermore, further research are needed for detection the SARS-CoV-2 in solid and biomass produced from WWTP processes due to flaks formation, followed by sedimentation in order to better understand the wastewater-based epidemiology and preventive measurement for other epidemics probably encountered in the future.

Detection of HEV RNA Using One-Step Real-Time RT-PCR in Farrow-to-Finish Pig Farms in Bulgaria.

(1) Background: HEV is a zoonotic, foodborne pathogen. It is spread worldwide and represents a public health risk. The aim of this study was to evaluate the presence of HEV RNA in farrow-to-finish pig farms in different regions of Bulgaria; (2) Methods: Isolation of HEV RNA from pooled samples of feces was performed using a QIAamp® Viral RNA Mini Kit followed by HEV RNA detection using a single-step real-time RT-PCR with primers and probes targeting the ORF 3 HEV genome; (3) Results: HEV RNA was detected in 12 out of 32 tested farms in Bulgaria (37.5%). The overall percentage of HEV-positive pooled fecal samples was 10.8% (68 of 630 samples). HEV was detected mostly in pooled fecal samples from finisher pigs (66/320, 20.6%) and sporadically from dry sows (1/62, 1.6%) and gilts (1/248, 0.4%); (4) Conclusions: Our results confirm that HEV circulates in farrow-to-finish pig farms in Bulgaria. In our study, we found HEV RNA in pooled fecal samples from fattening pigs (4-6-months age), shortly before their transport to the slaughterhouse indicating a potential risk to public health. The possible circulation of HEV throughout pork production requires monitoring and containment measures.

SARS-CoV2 mutations and impact on mortality: observational study in a sub-saharan Africa hospital

SummaryBackgroundOne year after the coronavirus disease 2019 (COVID-19) pandemic, the focus of attention has shifted to the emergence and spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs). The aim of the study was to assess the frequency of VOCs in patients followed for COVID-19 at Kinshasa university hospital (KUH) during the 3rd and 4th waves of the pandemic in Kinshasa. Hospital mortality was compared to that of the first two waves.MethodThe present study included all patients in whom the diagnosis of SARS-CoV-2 infection was confirmed by the polymerase chain reaction (PCR). The laboratory team sequenced a subset of all SARS-CoV-2 positive samples with high viral loads define as Ct < 25 to ensure the chances to generate complete genome sequence. RNA extraction was performed using the Viral RNA Mini Kit (Qiagen). Depending on the platform, we used the iVar bioinformatics or artic environments to generate consensus genomes from the raw sequencing output in FASTQ format.ResultsDuring the study period, the original strain of the virus was no longer circulating. The Delta VOC was predominant from June (92%) until November 2021 (3rd wave). The Omicron VOC, which appeared in December 2021, became largely predominant one month later (96%) corresponding the 4th wave. In-hospital mortality associated with COVID-19 fell during the 2nd wave (7% vs. 21% 1st wave), had risen during the 3rd (16%) wave before falling again during the 4th wave (7%) (p < 0.001).ConclusionThe Delta (during the 3rd wave) and Omicron VOCs (during the 4th wave) were very predominant among patients followed for Covid-19 in our hospital. Contrary to data in the general population, hospital mortality associated with severe and critical forms of COVID-19 had increased during the 3rd wave of the pandemic in Kinshasa.

Establishment and evaluation of a method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples

To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers' urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers' urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.

Evaluation of extraction and enrichment methods for recovery of respiratory RNA viruses in a metagenomics approach

Viral metagenomics is increasingly applied in viral detection and virome characterization. Different extraction and enrichment techniques may be adopted, however, reports on their effective influence on viral recovery is often conflicting. Using a three step enrichment steps, the effect of three extraction kits and the influence of DNase treatment with or without rRNA removal for respiratory RNA virus recovery from nasopharyngeal swab samples was evaluated. The viral cocktail containing six different RNA viruses pooled in equal volume were subjected to the different extraction and enrichment methods, sequenced using the Illumina MiSeq, and analysed using Genome Detective. The PureLink® Viral RNA/DNA Mini Kit (PureLink) was highly efficient with better recovery of all the viral agents in the cocktail. The use of rRNA treatment resulted in increased viral recovery with PureLink and QIAamp® Viral RNA Mini kit, while having comparable recovery rate as DNase only with the QIAamp® MinElute Virus Spin Kit. The observed low reads and genome coverage of some of the viruses could be attributed to their low abundance. Depending on sample matrix, extraction choice and enrichment strategy may influence recovery of respiratory RNA virus in metagenomics studies, therefore individual evaluation and adoption may be necessary for a robust result.